IE45416B1 - Improvements in or relating to ph-indicator compositions having controlled change of colour and ph on freezing and amethod for the preparation thereof - Google Patents

Abstract

PH indicator composition has controlled change of color at a selected sub-freezing temperature. The composition comprises at least one pH indicator, a buffer solution containing at least one inorganic ion in solution and a control agent or a precursor of a control agent. The buffer solution has a pH capable of changing when the temperature of the buffer solution is lowered to below freezing. The control agent is capable of effecting a change in ionic strength and proton composition of the frozen pH indicator composition to thereby control the temperature at which the color change occurs. A method of using the composition comprises lowering the temperature from a first temperature above freezing to a second temperature at which the pH indicator composition is in a substantially frozen state. The composition is particularly useful in a color change thermometer.

Description

The present invention generally· relates to an aqueous pH-indicator composition having controlled change of colour on freezing or at a selected sub-freezing temperature. The invention further relates to a method for preparing such a composition having controlled change of colour on freezing or at a selected sub-freezing temperature. According to some preferred embodiments, the invention more particularly aims at taking advantage of such a control of the colour change in order to obtain new compositions for temperature or time10 temperature indication, in the range from the freezing point of the composition down to about -90°C or below. Freezing in the present context implies the transition of the composition from liquid to solid state or suspension as opposed to supercooling which may occur in some instances when the tempe15 rature is lowered. In order to prevent supercooling, it is possible to add to the composition certain inorganic salts, such as KC1, NaCl etc. Further, it should be pointed out that although aqueous systems are preferred for the invention, it is not restricted to the use thereof but is also applicable to non-aqueous systems or mixtures of aqueous and non-aqueous systems. pH-indicators, which, in short, may be described as consisting of an acid-base couple where the acid has a colour different from that of the base, are generally used to deter25 mine or at least to give an idea of the pH-values in solutions A limitation of such pH-indicators is that they cannot give an exact determination of the pH-value since they have a transition interval for'the colour change from acid to base form which amounts to 1-2 pH units. This is one reason why in recent years the pH-indicators have increasingly been replaced by electrical pH-meters measuring the pH value of a solution more accurately. A still more serious limitation common to the pH-indicators and the pH-meters is that they do not allow measurement of the pH-values of frozen solutions, i.e.. that are in the solid state.

The reason why pH-indicators cannot generally be used to indicate the pH of frozen solutions is that the colour of the pH-indicator is not stable but prone to change when the solution freezes.

In conjunction with the present invention, the occurrence of this colour change upon freezing has been investigated and it has been found that it usually seems to take place from base colour to acid colour. Thus, if the solution has a pH close to or immediately above the transition interval of the pH-indicator (i.e. on the alkaline side of the transition interval), there is obtained a colour change from base colour in the liquid solution to acid colour in the frozen solution.

A prerequisite for the colour change is that the solution contains a substance which contributes not only to the ionic strength of the solution but also to the H+ : OH* ratio thereof.

Such substances are buffers which yield at least one inorganic ion and which may be -5 selected from inorganic buffers, such as phosphate buffer, and salts of organic acids. In buffer systems comprising a base moiety stronger than the acid moiety (either in the form of a stronger base ion or quantitatively more base ions than . . acid ions, e.g. an alkali salt of an organic acid, basic amino acids or other amines, amides or amino compounds), the colour change upon freezing may take place in the opposite direction, i.e. from acid colour to base colour.

Another problem which, seemingly, is not associated with the above-mentioned problem, is that there has been no simple and efficient device available hitherto to permit visual indication, in a readily perceivable fashion, of temperature or temperature-time conditions, e.g. temperature distribution 1θ in a freezing compartment. Thus, in order to record the temperature in for instance a freezer storage or in freezers or refrigerated boxes, use has hitherto been made of conventional thermometers or of temperature-sensitive electric means. These known devices for measuring the temperature, however, suffered from the drawback of sensing the temperature only at a single point in the compartment concerned. Consequently, no general picture is obtained o? the temperature conditions prevailing in the compartment and, what is more, the picture obtained may be misleading, if the sensing means, as if often the case, is disposed in the coldest part of the compartment, for instance adjacent the supply opening for cooling air in a cooling compartment.

The present invention aims at presenting a solution to the above discussed problem associated with the limited applicability of pH-indicators to frozen solutions, and at ensuring indication of pH values of both liquid and frozen solutions.

In a particularly preferred aspect of the invention. & 43416 the object thereof is also tc obtain a solution to the problem associated with indicators which, in a readily perceivable fashion, visually indicate for instance the temperature conditions of a material portion of a compartment.

In view of the above, it has been made possible, according to the present invention, to solve the above discussed problems in conjuntion with colour indication in the frozen state, by adding, to a pH-indicator composition containing a pH-indicating substance and a buffer eolution containing at least one inorganic ion in solution, a control agent which brings about a change in both the ionic strength and the H+ : OH” ratio of the total composition in the frozen state. This also prepares the ground for providing an indicator permitting simple visual temperature indication, as will be explained in greater detail hereinbelow.

According to the present invention, there is provided a pH-indicator composition having controlled change of colour at the freezing point of the composition or at a selected subfreezing temperature thereof, said composition being characterized by: (a) at least one pH-indicator in an amount sufficient to display a change of colour upon change of pH; (b) a buffer solution containing at least one inorganic ion in solution, wherein said buffer solution has a pH which is capable of changing by 2 to 5 pH units when said buffer solution is brought from the liquid to the frozen state; and (c) a control agent or a precursor of a control agent, wherein said control agent is capable of effecting a change in the ionic strength and H+ : OH” ratio of said pH-indicator composition when frozen thereby to cause, upon freezing, a shift in change in the pH of said pH-indicator composition from that due to the pH change of said buffer solution; - 5 4S416 and being further characterized in that said control agent is in an amount sufficient so that said pH-indicator composition is capable of exhibiting the same colour throughout the temperature range from above the freezing point of the composition to said freezing point or sub-freezing temperature where the colour change occurs.

Further, according to the invention, there is provided a method for prepa.ing a pH-indicator composition having controlled change of colour at the freezing point of the composition or at a selected sub-freezing temperature thereof said method being characterized by providing: (a) at least one pH-indicator in an amount sufficient to display a change of colour upon change of pH, (b) a buffer solution containing at least one inorganic ion in solution, wherein said buffer solution has a pH which is capable of changing by 2 to 5 pH units when said buffer solution is brought from the liquid to the frozen state; and adding to (a) and (b) (c) a control agent or a precursor of a control agent, wherein said control agent is capable of effecting a change in the ionic strength and H :0H ratio of said pH-indicator composition when frozen thereby to cause, upon freezing, a shift in change in the pH of said pHindicator composition from that due to the pH change of said buffer solution; and being further characterized in that said control agent is added in an amount sufficient so that said pH-indicator composition is capable of exhibiting the same colour throughout the temperature range from above the freezing point of the composition to said freezing point or sub-freezing temperature where the colour change occurs.

The composition according to the invention may also contain preservatives, such as n-butanol, as optional but preferred additives.

As indicated by way of introduction, the invention is not restricted to aqueous systems, although these are preferred. In addition to aqueous systems, it is possible to use alcohols, such as methanol, ethanol and dodecanol. Of course, the indicator substance, the buffer and the control agent must be soluble in the selected medium.

Without relating the invention to any specific theory, it is believed that the above described colour change of the pH-indieatqr upon freezing is due to the proton composition being radically changed upon freezing, said change being made up of two parts. Firstly, the total conductivity decreases upon freezing of the solution because of the substantially decreased mobility of the different ions in the solution, including H+ and OH-. Secondly, the mobility or availability of OH- is believed to decrease more than that of H+, that is an (apparent) increase in the ratio H+ : OH* (the proton composition) is produced upon freezing of the solution. This means that the frozen solution has an (apparently) lower or more acidic pH-value than the liquid solution. As the ' colour condition of the pH-indicator depends precisely on the 20 ratio H+ : OH- (i.e. the pH), the colour condition of the pHindicator will thus change from base colour to acid colour, provided that the change in the ratio H+ s OH- is sufficient for the transition Interval of the pH-indicator to be passed when the solution freezes. Investigations have shown that the change in the ratio H : OH upon freezing generally corresponds to a shift of the pH-value towards the acidic side by about 2 to 5 units depending upon the pH-indicator used. Thus, if the original, liquid solution is made more alkaline,or has from the 416 very beginning a pH about 2 to 5 units above the pK-value of the pH-indicator, the indicator will not change its colour upon freezing of the solution.

The above suggested explanation of the colour change of the pH-indicator upon freezing thus implies that the pH of the solution shifts towards a more acidic value. Because the solution has to be made more alkaline to compensate for such decrease in pH on freezing, the pH cf the liquid solution is shifted towards a more alkaline value. This may be considered as an apparent shift of the pK-value (transition interval) of the pH-indicator towards a more alkaline value because-of the freezing, and this consideration will be used e.g. in the following Examples.

In accordance with what has been discovered in conjunction with the present Invention, the colour change of the pH-indicator seems mainly to be dependent on the transition from liquid phase to solid phase only and to be substantially independent of the temperature, i.e. once the colour of the indicator has shifted because of the freezing of the solution, no further colour change occurs on subsequent lowering of the temperature below the freezing point.

It has also been established that the concentration of the substance (inorganic buffer or salt of an organic acid) in the solution that contributes to the ionic strength and H+ : OH ratio of the solution has such an effect that the higher the concentration of the substance (buffer) is in the solution, the more acidic the solution will become when it freezes.

As mentioned above, the problem associated with the shift of the transition interval on freezing may be solved according to the invention, by the addition of a control agent which causes a change in the ionic strength and the proton composition of the frozen solution and which, more particularly, counteracts the reduction of the conductivity as well as the X _ change of the ratio H : OH (i.e. the pH-value) of the solution on account of the freezing. It should be noted that the control agent need not itself change the h+ : OH ratio, the important'thing being, however, that the effect of the control agent is such that the H+ ; OH ratio of the frozen solution is adjusted. This effect can be achieved by using various control agents which act, for instance, by changing the pH of the solution, or by having a surfactant activity. According to the invention, it has been found that alcohols and carboxylic acids, in particular, are efficient as control agents of the above type. Here, by alcohols is meant organic compounds containing hydroxyl groups, and more particularly monovalent as well as polyvalent alcohols, e.g. methanol, ethanol, various glycols, glycerol and carbohydrates. 2θ Similarly, the carboxylic acid control agents according to the invention include organic compounds containing carboxyl groups, and more particularly both mono-, di- and polycarboxylic acids which in turn, in addition to the ordinary” aliphatic carboxylic acids, comprise aromatic carboxylic acids, keto acids, •5 hydroxy acids and amino acids (including the amides and salts of these acids).

In practice, the pure carboxylic acids are not used as control agents since this would bring about an unwanted lowering of the pH of the solution, but before the carboxylic acid control agent is added to the solution, it is adjusted, e.g. by addition of NaOH, to the same pH as the solution.

This implies that the added carboxylic acid control agent substantially corresponds to a solution of the free acid and its salt. Alternatively and preferably, a salt of the acid (suitably an alkali metal or ammonium salt) may be used directly as carboxylic acid control agent, the adjustment of pH being effected with e.g. HC1. 1θ According to the invention, it is also possible, in order to change the ionic strength and the proton composition, to use, as a control agent, a precursor substance, i.e. a material capable of producing a substance which changes the ionic strength and the H+ : OH ratio. As an example of such precursor substances may be mentioned an enzyme-substrate mixture which, by the action of the enzyme on the substrate, produces the required substance.

The above-mentioned control agents are suited for systems that become more acidic when freezing (solidifying).

As a general rule, a control agent for a system which becomes more acidic when freezing, is a substance comprising an ion from a strong base and an ion from a weak acid, while a control agent for a system which becomes more alkaline when freezing, is a substance comprising an ion of a weak base and an ion of a strong acid. It should be noted that alcohols may be used as control agents for both types of systems.

The exact mechanism of the control agent according to the invention has not been clearly established, but with e.g. 4341 glycerol it is believed that this control agent acts by being capable of attracting water molecules, probably together with the pH-indicator, which makes the pH-indicator maintain its colour. Further, glycerol has a strong proton bonding ability, which produces a decreased mobility or accessibility of H+ as compared with 0H~, i.e. an (apparently) more alkaline pH-value This shift of the pH-value towards a more alkaline pH-value counteracts the shift of the pH-value towards a more acidic value which appears in the frozen solution without addition of control agent, as explained earlier. Because of its proton bonding ability, glycerol thus (apparently) changes the pHvalue of the frozen solution towards a more alkaline value.

The carboxylic acid control agents of the present invention bring about themselves an actual change in the con15 ductivity, i.e. both in the total conductivity and in the mutual ratio H+ : OH . Depending on whether the selected carboxylic acid control agent cooperates with the particular buffer system or not, the shift of the transition interval of the pH-indicator upon freezing may be either counteracted or further accentuated.

In the use of the above mentioned control agents, it has been noticed that they have the unexpected effect of making the colour change of the pH-indicator upon freezing temperature-dependent, i.e. the colour change need not occur directly in connection with the freezing, but may be caused to occur at temperatures below the freezing point of the solution. This applies to the alcohol control agents when an inorganic buffer or a salt of an organic acid is present, while it may occur for the carboxylic acid control agents (including acid salts) also in the absence of an inorganic buffer (carboxylic acid plus alkali salt of the carboxylic acid themselves form a buffer system)'. Furthermore, it has been discovered that the θ· temperature dependency of the colour change of the pH-indicator is dependent upon the concentration of the added control agent and upon the initial pH of the liquid solution. Thus, an increasing concentration of control agent gives a decreased with colour change/ temperature at a constant pH, while a more alka10 line initial pH of the liquid solution gives a decreased tempe rature for the colour change at a constant concentration of the control agent. Xt has hereby become possible to obtain (besides that the colour change of the pH-indicator is counter acted, when freezing is effected, by the addition of a control agent) a colour thermometer which, due to the fact that the colour change of the pH-indicator has been made temperaturedependent, displays one colour above the transition temperature (base colour) and another colour below the transition temperature (acid colour). This colour change can be made very distinct and perceptible such that the colour thermometer will give clear information of such small temperature differences as 1-2°C. By filling an elongated tube with a composition of the above-mentioned type according to the invention, there is thus provided an elongated colour thermometer which, if 2θ arranged on the wall in a cooling or freezing room, will show below what level the temperature is satisfactorily low and above what level the temperature is too high.

Instead of filling an elongated tube with such 43416 solution, as indicated above, it is possible to produce an elongated strip with regularly spaced transparent chambers, each filled with the solution. This embodiment is more practical than the embodiment comprising an elongated tube, because the strip may easily be severed between the chambers to produce strip portions of the desired length. Furthermore, the chambers may be filled with different solutions showing colour change at different temperatures below freezing point.

In this way, a thermometer is produced which by its colour displays the prevailing temperature in a readily perceptible and visually distinct way. A further application of the colour thermometer described above is to produce a single chamber or capsule filled with such solution which gives a colour change at a certain temperature, e.g. -18°C, and attach the capsule to a deep-frozen product. At each phase of the handling of the deep-frozen product, the capsule enclosing the indicator solution will then show if the temperature is above or below the chosen temperature to be indicated (e.g. -18°C). It should be noted that each separate colour thermometer gives an indication cf colour change only at a particular temperature which is characteristic of the selected combination of indicator solution, pH-value and content of control agent.

The pH-indicator'is typically incorporated in an amount of from about 0.01 to 0.05% by weight/volume. However, by suitably com25 bining these factors, it is possible to provide a colour thermometer which displays a colour change at an optional temperature. It should also be emphasized that the above described colour thermometer according to the present Invention is reversible, i.e. it may be used again in order to indicate temperature changes beyond the particular colour temperature that is specific to the chosen colour thermometer.

To facilitate understanding of the invention, a number of illustrative examples thereof are given below.

Example 1 This example illustrates the colour changes in two different pH-indicator substances, nitrazine yellow and bromthymol blue, when freezing is effected. The indicator solutions were buffered with 0.02 M Ha2HPO4 (pH = 9.4). The pH-value was adjusted with 1 N HC1. The results are shown in Table 1.

As will clearly appear from Table 1, both nitrazine yellow and bromthymol blue in a solution which is buffered with the inorganic buffer Na2HPO4 indicate a shift of the transition interval when freezing is effected.

By way of comparison, corresponding tests were carried out involving, however, indicator solutions which were buffered with 0.02 M of the organic buffer tris-hydroxymethyl aminomethane (Tris). Here, no appreciable shift of the transition interval was obtained, which was to be expected since Tris is a substantially non-dissooiated organic compound which contributes only insignificantly to the proton conductivity.

In order to demonstrate that it is not the degree of dissociation that is of importance whether or not the transition interval is to shift, tests were also carried out using indicator solutions which were buffered with Tris-buffers and to which had been added NaCl, i.e. a substance changing only the ionic strength but not the H+ : OH ratio. As is appa14 rent from Table 1, no appreciable shift of the transition interval was obtained in this instance either.

Example 2 In this example, tests were carried out using a large number of different pH-indicator substances, and it was found that all of them displayed a shift of the transition interval on freezing. Table 2 is a compilation of the different pHindicators tested, their normal transition intervals at +20°C (solid lines) and at -20°C (broken lines). In all instances, the indicator solutions were buffered with 0.02 M NajHPO^. pH was adjusted with 1 N HCl or 1 M NaOH.

Example 3 In this example, tests were conducted involving the addition of glycerol to different pH-indicator solutions which were buffered with phosphate buffers, as described above. The measurements were effected at +20°C and at -18°C, the pH-value being maintained constant at 7.0. All samples were frozen at -18°C. The results will appear from Table 3. It should be pointed out that the indicated glycerol concentrations refer to a 87% glycerol solution.

Example 4 This example shows the effect of pH, temperature and glycerol content on the colour change of bromcresol green.

The indicator solution contained, as above, 0.02 M Na2HPO4· The results will appear from Table 4.

Table 1 0.02 M Na„lll’O, buffer 2 4 pH 0 (+20C) Nitrazine yellow colour at +20°C -20°C Bromthymol blue colour at + 20°C -20°C 9.40 dark violet blue yellow 9.00 If greenish brown It II 8.50 II yellowish red tl II 8.00 II if II II 7.50 »1 II bluish green If 7.0Q violet II green If 6.50 brown II It II 6.00 brownish red tr yellowish green fl 5.50 reddish yellow II yellow ft 5.00 yellowish red It ir It 4.60 II fl ll It pH 0 (+20 C) Nitrazine yellow colour at + 20°C 0.02 M Tris-buffer Bromthymol ' blue colour at + 20°C Oa -20 6 -20°C 9.40 dark violet dark violet blue blue 9.00 It ft II II 8.50 It 11 It bluish green 8.00 II ft II green 7.50 It bluish green greenish yellow 7.00 violet violet green yellow 6.50 brown light violet II II 6.00 brownish red yellowish brown yellowish green II 5.50 reddish yellow yellowish red yellow II 5.00 yellowish red II It II 4.60 II It If It 43413 0.02 M Tris-buffer + 0.1 Ji NaCl PH (+20°C) Nitrazine yellow colour at + 20°C -18°C Bromthymol blue colour at + 20°C -18°C 9.4 violet violet blue light blue 9.0 II II II II 8.5 If II It II 8.0 II It tl II 7.5 II II greenish blue bluish grey 7.0 light violet II green greyish green 6.5 lilac brown It II yellowish grey 6.0 brown light brown yellowish green yellow 5.5 - - yellow II 5.0 yellow yellowish red II It 4ΰ4ΐθ CM Φ «Η tf Η & Ο *0 r-i ϋ W Ρ Φ •tf rC Φ tf P •d «rt » & 4J •rt & o *j Φ •tf O r-i Φ rH Tj rt rt rt o Φ rH φ 0 •rt rt φ X«rt > 11 is H > U M PH P rt S >» > > β Ο rt ίϊ β Χ> Ο > Γ-4 Ο ΛΗ μ μ m λ •rt >, 5S 4J Φ JJ 43 Φ rt 43 63 60 ω μ·η c ϊ -ri Η Ο Ό u *-j η μ φ 49 κ Ρ ο Ρ ρ ιι η 60 r-i ι—i Ο rt Ρ ο tf υ «Η •tf β •rt I Β α Φ Ο β «Η ·Η rH rH φ Φ r-t X « rrt J* Ο Φ 44 S β 43 r*» *rt 0« N 1-4 u tf o ShB O 44 φ rt ·Η 43 S Pm •tf Φ rt O β Φ JS Φ β r4 •tf Φ P« Φ Φ • rt rt •ϋ rt β φ Φ 60 tf. •rt rt 60 0 β φ rrt 1—4 β irt tf •tf •tf β 0 0 •tf Φ Ο rt Φ Φ rH tf tf Φ 43 β ο Ρ Ρ 49 Φ Φ rt β. φ rt rt Ο Λ rH •rt rrt ι—< CJ CJ o rt Ο. is >» Ο Ο 6 £ 60 ο £ •α •c tf S o o β 1—1 ο 44 4> Φ is lu rt 0 45 rt Φ Φ Ρ 43 P9 m o ο 09 2 2 ο ΕΗ o «rt > x: co β« gp ? s O ϋ tf Φ tf rH β rH X3 43^ rH 43 H o •tf H > H ϋ B rt iH P |j •tf »tf »tf »tf ej LO Table Table 4 Temperature °C pH 4.0 0.52 glycerol (1 pH 5 .0 1.02 glycerol pH 6.0 0.52glycerol -14 Bluish green Bluish green Bluish green -16 Yellow Greenish yellow Yellowish green -18 Yellow Yellowish green Yellowish greenj -20 Yellow Yellowish green Yellow -22 Yellov Yellow Yellow Example 5 This example shows the effect of pH, temperature and glyce10 rol content on the colour change of chlorophenol red. The indicator solution contained, as above, 0.02 M NajHPO^. The results will appear from Table 5.

Table 5 Temperature °C pH 5.0 1.02 glycerol pH 5.5 0.52 glycerol pH 6.0 0.52 glycerol -14 Violet Violet Violet -16 Yellowish red Red Yellow -18 Yellowish red Yellow Yellow -20 Yellow Yellow Yellow -22 Yellow Yellow Yellow «5416 Table 6 Control agent (weight/volume 2) Chlorophenol red Bromcresol + 20°C green -18°C + 20°C -18°C 0 = Check (only phosphate buffer) Red Yellow Blue Yellow Dextrose (52) Red Yellowish red Blue Yellowish green ! Sorbitol (52) Red Yellowish red Blue 1 Yellowish’ green Saccharose (52) 1 I. Red Yellowish red Blue 1 Greenish ( yellow Tween 80 (surfactant containing polyoxyethylene (20) sorbitan monooleate (10%) Reddish yellow Yellowish red Green Green (Glycerol (8.7%) Red Red Blue Blue Ethylene glycol (10%) Red Red Blue Blue Polyethylene glycol (5%) Red Red Bluish Breen Blue Cellosolve (5%) Red Red Bluish green Blue Methanol (5%) Red Yellowish red Bluish green Green Ethanol (5%) Red Yellowish red Bluish green Blue Sodium glutamate (5%) Red Red Bluish green Blue Sodium succinate (5%) Red Yellowish red Bluish green Green Sodium acetate (5%) Red Red Bluish green Light green Sodium citrate (5%) Red Red Bluish green Green Ammonium formate (5%) Red Red Bluish green Blue .Example 6.

This example illustrates the colour-change counteracting effect of different control agents according to the invention.

The indicator solutions consisted respectively of chlorophenol red and bromcresol green in aqueous solution buffered with 0.02 M NajHPO^. The pH values of the solutions were adjusted with HCl to 6.0-6.5. The results will appear from Table 6.

Example 7 This test demonstrates a) the shift of the transition inter-K val in a pH-indicator (Congo red) on freezing; and b) that this shift does not continue upon continued temperature reduction below freezing point, i.e. as long as the?indicator composition is in either the liquid or the solid phase, the transition interval remains independent of temperature, provided no control agent according to the invention, e.g. glycerol, has been added. The results will appear from Table 7.

Table 7 The colour of a Congo-red indicator in an aqueous solution buffered With 0.02 M NaoHP0,. 2 4 pH (+20°C) -20°C -15°C ~ 6°C +5°C + 10°C +20°C 9.9 red red red red red red 9.15 It II ,1 It II n 8.5 II II reddish brown II II 8.0 black black black II II II 7.5 II II II II It II 7.0 π II II It II It 6.5 II II II II II II 6.0 II II II II It It V \....... --y,-Solid Liquid Example 8 In order to demonstrate that the carboxylic acid control agents according to the invention may cause the transition interval of the pH-indicator to be temperature-dependent in the frozen system also in the absence of a buffer, tests were carried out using a solution containing 5% sodium succinate (carboxylic acid control agent), and chlorophenol red as pH-indicator. The colour of the indicator was observed at +20°C as well as at three different temperatures below freezing point, the results being as follows: Temperature (°C) +20 Colour red red yellowish red yellowish red The colour change occurring between -6°C and -10°C is indicative of a change of the transition interval of the pH-indicator.

Example 9 Table 8 shows various examples of enzyme-substrate-combinations which are precursors to the desired control agent and. which produces the desired control agent by enzymatic reaction. It will be appreciated that the combinations shown in Table 8 constitute illustrative examples only of usable precursors and that one skilled in the art can select many other enzyme-substrate-combinations for producing control agents by enzymatic reaction. <*3416 Table 8 Enzyme Substrate Control Agent Other Products Phosphatase Phosphatase Esterase Lipase Glucose-6-phosphate β-glycerophosphate Ethyl Butyrate Triglyceride Glucose Glycerol Ethanol Glycerol Phosphate Phosphate Butyrate Salt of fatty acid Example 10 A composition for use as the colour thermometer of the invention is obtained in the following way. 350 ml of 99.5% glycerol, 4 1 of a 0.5% solution of chiorophenol red and 800 ml of n-butanol (preservative) were added to 2 1 of an aqueous solution containing 142.5 g of NajHPO^ · 2^0, the resulting mixture being then diluted with distilled water to a total volume of 40 1« The solution obtained was adjusted with 1 N. HC1 to pH 7.5.

The solution was then poured into transparent chambers in a strip, as described above. This resulted in a colour thermometer which upon testing displayed violet colour at temperatures above about -18°C and yellow colour at temperatures below about -18°C.

Example 11 1 of the colour thermometer solution of Example 10 was modified by the addition of 35.6 g of NajHPO^ · 2^0 and 50 ml of 99.5% glycerol. The pH of the solution was 7.96.

When using this solution as a colour thermometer in the above-defined fashion, a change from violet to yellow was recorded when the temperature was lowered to below about -24°C .

Claims (31)

1. A pH-indicator composition having controlled change of colour at the freezing point of the composition or at a selected sub-freezing temperature thereof, said composition 5 being characterized by (a) at least one pH-indicator in an amount sufficient to display a change of colour upon change of pH, (b) a buffer solution containing at least one inorganic ion in solution, wherein said buffer solution has a pH which is 10 capable of changing by 2 to 5 pH units when said buffer solution is brought from the liquid to the frozen state, and (c) a control agent or a precursor of a control agent, wherein said control agent is capable of effecting a change in the ionic strength and H + : 0H‘ ratio of said pH-1ndicator composition 15 when frozen thereby to cause, upon freezing, a shift in change in the pH of said pH-indicator composition from that due to the pH change of said buffer solution; and being further characterized in that said control agent is in an amount sufficient so that said pH-indicator composition is capable of exhibiting the same 20 colour throughout the temperature range from above the freezing point of the composition to said freezing point or sub-freezing temperature where the colour· change occurs.

2. A pH-indicator composition as claimed in claim 1, wherein the buffer solution is an aqueous solution. 4 5416

3. A pH-indicator composition as claimed in claim 1 or 2, wherein said pH-indicator has a pK-value in said pHindicator composition and said control agent is in an effective amount to cause a change in said H + : OH ratio so that 5 (a) for a pH-indicator composition which becomes more acidic on freezing, said proton composition is changed to a value less than at said pK-value, and (b) fcr a pH-indicator composition which becomes more alkaline on freezing, said H : OH ratio is changed to 10 a value greater than at said pK-value.

4. A pH-indicator composition as claimed in claim 1, wherein the control agent is an alcohol or a precursor therefor.

5. A pH-indicator composition as claimed in claim 4, wherein the control agent comprises at least one hydroxyl group 15 ' and is selected from the group consisting of methanol, ethanol, glycols, glycerol and carbohydrates.

6. A pH-indicator composition as claimed in claim 5, wherein the carbohydrate is selected from the group consisting of saccharose. Sorbitol and dextrose.

7. A pH-indicator composition as claimed in claim 3, wherein the control agent, for a pH-indicator composition which becomes more acidic on freezing, is a substance comprising an ion from a strong alkali and an ion from a weak acid.

8. A pH-indicator composition as claimed in claim 7, >g wherein the control agent is a salt of a carboxylic acid.

9. A pH-indicator composition as claimed in claim 8, wherein the carboxylic acid moiety is derived from a carboxylic acid comprising at least one carboxyl group and is selected from the group consisting of aliphatic and aromatic carboxylic 5 acids, keto acids, hydroxy acids and amino acids.

10. A pH-indicator composition as claimed in claim 9, wherein the carboxylic acid is selected from the group consisting of for.nic acid, acetic acid, succinic acid, citric acid and glutamic acid. θ

11. A pH-indicator composition as claimed in claim 3, wherein the control agent, for a pH-indicator composition which becomes more alkaline on freezing, is a substance comprising an ion of a weak alkali and an ion of a strong acid.

12. A pH-indicator composition as claimed in claim 1, ’ wherein the precursor of the control agent is an enzyme-substrate mixture which produces the control agent by enzymatic reaction.

13. A pH-indicator composition as claimed in claim 12, wherein the precursor is selected from the group consisting of ' phosphatase/glucose-6-phosphate, phosphatase/B-glycerophosphate, esterase/ethyl butyrate and lipase/triglyceride.

14. A pH-indicator composition as claimed in claim 1, wherein said composition is an aqueous composition which comprises (a) chlorophenol red as the pH-indicator (b) Na 2 HPO 4 as the buffer, and (c) glycerol as said control agent. •j a a ι ο -

15. A pH-indicator composition as claimed in claim 14, wherein each litre of liquid composition comprises about: 50 ml 0.4 M aqueous solution of Na 2 HPO 4 · 2H 2 O 100 ml of 0.5% chlorophenol. solution 5 9 ml of glycerol (99.5%) 20 ml of n-butanol as an optional preservative distilled water to make 1 litre and 1 M HCl to adjust the pH to 7.5.

16. A pH-indicator composition as claimed in claim 15, θ wherein to each litre of said liquid composition is further added 3.56 g Na 2 HPO 4 · 2H 2 O and 5 ml glycerol (99.5%).

17. A method for preparing a pH-indicator composition at the. point of the composition having controlled change of colour ireezing^or at a selected thereof sub-freezing temperature^as claimed in any one of the preceding claims, said method being characterized by providing (a) at least one pH-indicator in an amount sufficient to display a change of colour upon change of pH, (b) a buffer solution containing at least one inorganic ion in solution, vzherein said buffer solution has a pH 0 which is capable of changing by 2 to 5 pH units when said buffer solution is brought from the liquid to the frozen state, and adding to (a) and (b) (c) a control agent or a precursor of a control agent, wherein said control agent is capable of effecting a change in ’ the ionic strength and H + : OH ratio of said pH-indicator composition when frozen thereby to cause, upon freezing, a^ciiaAge in the pH of said pH-indicator composition from that due to the pH change of said buffer solution; 43413 and being further characterized in that said control agent is added in an amount sufficient so that said pH-indicator composition is capable of exhibiting the same colour throughout the temperature range from above the freezing point ofthe comnosition to said freezing point or sub-freezing temperature . where the colour change occurs.

18. A method as claimed in claim 17, wherein said control agent is added to an aqueous buffer solution.

19. A method as claimed in claim 17 or 18, wherein said control agent is added in an effective amount to cause a change in said H ·' θΗ ratio so that: (a) for a pH-indicator composition which becomes more + acidic on freezing, said H : OH ratio is changed to a value less than at · the pK-value of the pH-lndicator, and (b) for a pH-indicator composition which becomes more alkaline on freezing, said ,H + : OH ratio - is changed to a value greater than at tne pK-value of the pH-indicator in said pHindicator composition.

20. A method as claimed in claim 17, wherein aa said control agent there is added an alcohol or a precursor therefor.

21. A method as claimed in claim 20, wherein as said control agent there is added an alcohol which comprises at least one hydroxyl group and is selected from the group consisting of methanol, ethanol, glycols, glycerol and carbohydrates.

22. A method as claimed in claim 21, wherein as said control agent there is added a carbohydrate selected from the group consisting of saccharose, sorbitol and dextrose. 4S410

23. A method as claimed in claim 19, wherein the pH-indicator composition becomes more acidic on freezing, and as said control agent -there is added a substance comprising an ion from a strong alkali and an ion from a weak acid. 5

24. A method as claimed in claim 23, wherein as said control agent there is added a salt of a carboxylic acid.

25. A method as claimed in claim 24, wherein the carboxylic acid moiety is derived from a carboxylic acid comprising at least one carboxyl group and is selected from the group consisting of aliphatic and aromatic 10 carboxylic acids, keto acids, hydroxy acids and amino acids.

25. A method as claimed in claim 25, wherein the carboxylic acid is selected from the group consisting of formic acid, acetic acid, succinic acid, citric acid and glutamic acid.

27. A method as claimed in claim 19, wherein the pH-indicator

15. Composition becomes more alkaline on freezing, and as said control agent there is added a substance comprising an ion of a weak alkali and an ion of a strong acid.

28. A method as claimed in claim 17, wherein as said precursor of the controlagent there is added an enzyme/substrate mixture which

16. 20 produces the control agent by enzymatic reaction.

29. A method as claimed in claim 28, wherein the precursor is selected from the group consisting of phosphatase/glucose-6-phosphate, phosphatase/ -glycerophosphate, esterase/ethyl butyrate and lipase/ triglyceride .

17. 25 30. A pH-indicator composition having controlled change of colour at the freezing point of the composition or at a selected sub-freezing temperature thereof, substantially as hereinbefore described with reference to any one of the foregoing Examples.

31. A method as claimed in claim 17 and substantially as herein30 before described with reference to any one of the foregoing Examples. Dated this 10th day of May 1977 BY: TOMKINS & CO,,